In the field of molecular diagnostics, the amplification and detection of nucleic acids is of considerable significance. Examples for diagnostic applications of nucleic acid amplification and detection are the detection of viruses such as Human Papilloma Virus (HPV), West Nile Virus (WNV) or the routine screening of blood donations for the presence of Human Immunodeficiency Virus (HIV), Hepatitis-B (HBV) and/or -C Virus (HCV). Said amplification and detection techniques are also suitable for bacterial nucleic acid targets or the analysis of oncology markers or the like.
The most prominent and widely used method for amplification and detection of nucleic acid targets is the Polymerase Chain Reaction utilizing a polymerase enzyme (PCR, U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202). Related significant improvements are, e.g., real-time detection of amplified products during PCR utilizing modified oligonucleotides carrying reporter groups or labels known as hydrolization or 5′-nuclease probes such as used in commercial assays on COBAS® TaqMan® (U.S. Pat. No. 5,210,015 and U.S. Pat. No. 5,487,972). Other improved amplification and detection methods are known as Molecular Beacons technology (WO 95/13399) or methods utilizing an oligonucleotide comprising a minor groove binder (MGB) portion (WO 03/062445 and WO 2006/135765).
It is further known that the use of primers containing an added oligonucleotide with a high GC content at the 5′ terminus of at least one of these primers displays an improvement in amplification efficiency (Q. Liu et al., Genome Research vol. 7 (1997), p. 389-398; WO 01/94638; US 2004/0110182). The final quantity of the amplified product after approximately 12 to 40 cycles of PCR is markedly higher for primers to which e.g. a GGAC unit has been added to the 5′ termini than for the unmodified primers.
Afonina et al. (BioTechniques vol. 43(3) (2007), p. 1-3; WO 2006/135765) describe the increase of real-time PCR fluorescent signal and thereby obtaining improved amplification efficiency by using primers with short adenine and thymine rich flaps, scattered randomly, at the 5′ terminus and minor groove binder (MGB) fluorescent hybridization probes.
Furthermore, in some PCR assays, side products like the formation of high molecular weight products might have substantial impact on the amplification efficiency, e.g., creates false positive or false negative results. In particular for low titer samples a decrease or dropout of detection signals is expected due to the formation of high molecular weight amplification products leading to false negative results. The amplification efficiency is usually more reduced as more PCR cycles are carried out.
In quantitative PCR reactions often an internal validation and quantitation standard is added and is co-amplified. It controls the preparation and amplification processes and compensates for effects of inhibition. Formation of high molecular weight products during amplification of the internal standard may lead to suppressed signals and an invalid standard so that the PCR reaction which is controlled by the internal validation and quantitation standard is invalidated.
A source of false positive results is “carryover contamination”, where a PCR product (amplicon) from a prior PCR reaction contaminates subsequent PCR assays. The contaminant may be transmitted by a technician, an instrument or even via aerosol. High molecular weight products are more resistant than single amplicons to contamination prevention measures like uracil-DNA glycosylases (U.S. Pat. No. 6,713,294) and therefore significantly increase the risk of carryover contamination. In a true negative sample, where the target nucleic acid is absent, the contaminant creates a false positive result. In a true positive sample, where the target nucleic acid is present, the contaminant is co-amplified with the true target. Such co-amplification may distort a result of a quantitative assay, where the exact amount of the true target must be determined.
Thus, there is a need in the art to provide a method for simple and reliable amplification and detection of a nucleic acid target. There is in particular a need to provide an appropriate improved method with focus on suppressing high molecular weight by-products.